The Misuse of Most-Probable Number in ‘Rogue’ BI Remediation

By: James Agalloco Agalloco & Associates

Introduction

In recent years, the term ‘rogue’ has been used to explain away unexpected positive results with biological indicators exposed to ambient pressure vapor phase H₂O₂ processes.ⁱ¹ When failures occur during validation studies, one must consider whether the results are a consequence of process deficiencies, or in the experimental design itself. The complexity of vapor phase H₂O₂ processes (the unavoidable presence of two phases) and the uncertainties surrounding biological indicators (there is no established D-value methodology for them) are such that investigations are challenging. Vapor processes used for aseptic processing often have a dual objective – sterilization for product contact and indirect product contact parts and decontamination for the remainder of the system.^2,3

Dual phase vapor processes are substantially more complex than single phase sterilization methods like moist heat, dry heat and gas processes. The unavoidable bi-phasic nature of vapor phase delivery systems makes determination of sterilizing phase agent concentration nearly impossible.^ii4-6 Most users have found it challenging to delve deeply into vapor process details and focused their attention on the biological indicators. Increasingly the belief was that characteristics of the biological indicator were responsible for what are believed to be otherwise acceptable vapor phase processes to yield anomalous results. Explanations for the ‘rogue’ phenomena are numerous: excessive resistance, presence of soil, crevices in the substrate, clumping of spores and combinations of these. The use of multiple biological indicators in close proximity in combination and an adaptation of Most Probable Number (MPN) population estimation was adopted as a means for negating the positive results and accepting the process as is.^1,7,8

MPN was initially developed for the analysis of water samples especially those where the expected microbial population is very low (less than 5 CFU).^9,10 In this usage of MPN the results from multiple replicate samples are used to estimate the microbial population. The application of MPN practices to H₂O₂ processes appears to have been first suggested by Sigwarth and Mouitandat in systems where MPN was used to resolve recurring problems with biological indicators in vapor H₂O₂ systems.⁸ The use of MPN in this manner was included in PDA’s TR #51 as a means for addressing positive results.¹

The underlying assumption in the use of MPN for resolution of anomalous results is the existence of a log-linear death curve (and by inference a consistent D-value) which, while postulated by many practitioners, has never been established in a bi-phasic system. Numerous subsequent publications have made it clear that vapor phase H₂O₂ processes are always bi-phasic precluding the use of concepts derived from log-linear kinetics.^5,6,11-14 The use of MPN as a rationale for negating positive results is inappropriate in the absence of a linear death curve. The sage advice of Dr. Pflug relative to the greater importance of microbiological results in relation to physical data must be recalled: “the bugs don’t lie!”¹⁵ Anomalous bioindicator results should have redirected equal attention to the more complex resolution of the uncertainties within the vapor H₂O₂ delivery process.¹⁶

Most-Probable Number Misapplication

The workaround afforded using MPN in vapor phase H2O2 sterilization processes has other weaknesses that should be explored.

• In the majority of MPN studies for vapor processes there are only 3 replicate BIs, which is the minimum possible. The limited number of replicates weakens the utility of the results.
• MPN tests were initially intended for use in quantification of microbial populations from water using dilution samples with populations less than 5 CFU/ml. It is inappropriate to assign a population of 1 to a positive unit when it could be anywhere below the initial population (customarily >1 x106 CFU/indicator). 

In the rogue biological indicator work-around these flaws are assumed to be of no consequence. Positive results are ignored because when killing 2 out of 3 BI’s the log reduction is seemingly high enough. This ignores the possibility that it may not be averaging 0, 0, and 1, but rather 0, 0 and a positive result as high as 1x10⁶.

A Parallel Event Approached Very Differently 

Consider what occurs when a positive occurs in a validated process using other than vapor phase H₂O₂. An investigation would be immediately opened, and a thorough evaluation of the process initiated. The author participated in a lengthy investigation during the validation of a steam sterilization process where the delivered physical lethality in several items was less than expected compared to results with a nearly identical load containing many of the same items processed less than 2 months previously. We did not experience positive BI results, nevertheless, it was reviewed intensely. Release of affected products was suspended for several weeks, recall of other lots was considered, and repeat investigative and validation studies conducted .The root cause was identified after 6 weeks, and the issue corrected, but the efforts undertaken to resolve the anomalous results were substantial. Recognize that all of this was undertaken without a positive BI result on a long established and closely monitored process. Use of MPN to dismiss more serious non-conforming results on less well characterized and more complex processes using vapor H₂O₂ is minimally defensible in my opinion. None of the publications describing the use of MPN for resolution of biological indicator positives mention comparable attention given to investigation of the vapor sterilization process as root cause for the positives.^1,7,8,16

The Bottom Line

Vapor phase H₂O₂ process in many systems is decidedly non-uniform and may never reach a steady state condition (the exact opposite of what occurs with other sterilization process) and necessary for any assumption of reliable cycle efficacy. The prevalent MPN practice with ‘rogues’ is just an expedient means to move past the positive results and avoid the challenges associated with potential deficiencies in the vapor process being evaluated. 

References

1. PDA Technical Report No. 51: Biological Indicators for Gas and Vapor-Phase Decontamination Processes: Specification, Manufacture, Control and Use. PDA, Bethesda, Md, 2010.
2. EMA, Annex 1, Manufacture of Sterile Medicinal Products, 2022.
3. FDA, Submission and Review of Sterility Information in Premarket Notification (510(k)) Submissions for Devices Labeled as Sterile, January 2022.
4. Agalloco, J. & Akers, J., “Overcoming Limitations of Vaporized Hydrogen Peroxide - Hydrogen Peroxide – Highly Potent & Highly Problematic,” Pharmaceutical Technology. Volume 37, No. 9, pp.46-56, 2013.
5. Agalloco, J., & DeSantis, P., “The Science Behind Hydrogen Peroxide Decontamination and Sterilization,” published on-line on LinkedIn.com, January 2024 and American Pharmaceutical Review, Vol. 28, No. 1, pp. 30-36, 2025.
6. Agalloco, J., “Ridding the World of ‘Rogues’: Improving Vapor Phase H2O2 Sterilization and Decontamination Processes”, PDA Journal of Pharmaceutical Science & Technology, Vol 77, No. 5., pp 412-419, 2023.
7. Drinkwater, J., Chewins, J., & Steele, G., “Biological indicators don’t lie, but in sporicidal gassing disinfection cycles do they sometimes confuse the truth?”, European Journal of Parenteral & Pharmaceutical Sciences; Vol. 14, No. 1, pp 5-10, 2009.
8. Sigwarth, V., & Moirandat, C., Development and Quantification of H2O2 Decontamination Cycles. PDA Journal of Pharmaceutical Science and Technology, Vol. 54, No. 4, pp 286-304, 2000.
9. Standard Methods for the Examination of Water, Sewage and Industrial Wastes, APHA, AWWA, & FSIWA, 10th Edition, p. 385,1955.
10. Woodward, R.L., “How Probable is the Most Probable Number?,” Journal of American Water Works Association, Vol.49, pp 1060-1068, 1957. 
11. Watling, D., “Is H2O2 a Wet or Dry Process?”, Presentation at PDA Isolator Technology Conference, 2002.
12. D. Watling and M. Parks, “The Relationship Between Saturated Hydrogen Peroxide, Water Vapour and Temperature,” Pharmaceutical Technology Europe, 16 (3), 2004,
13. Unger-Bimczok, B., Kottke, V., Hertel, C. and Rauschnabel, J, “The influence of humidity, hydrogen peroxide concentration, and condensation on the inactivation of Geobacillus stearothermophilus spores with hydrogen peroxide vapor,” J Pharma Innovation, Vol. 3, pp. 123–133, 2008.
14. Agalloco, J., “Biological Indicators, Process Lethality and Vapor Phase Hydrogen Peroxide Processes,” PDA Journal of Pharmaceutical Science & Technology, Vol. 70, No. 5, pp 556-563.
15. Pflug, I. J. Microbiology and Engineering of Sterilization Processes,14th ed.; Environmental Sterilization Laboratory: Otterbein, IN, pp 683. 2010.
16. Sandle, T., “Rogue Biological Indicators: Are They A Real Phenomenon?,” Journal of Validation Technology, 26 (1), 2020.

ⁱThe focus of this publication is ambient pressure hydrogen peroxide processes have been given various identities: ‘vaporized H₂O₂’, VHP, VPHP, dry vapor, vapor fogging and others. They have also been described as either a ‘dry’ or ‘gas’ processes or as a ‘wet’ or ‘micro-condensed’ process.¹¹ The diversity of descriptive terms and the variety of system designs may have contributed to the uncertain outcomes some users have experienced.

ⁱⁱThere are other H₂O₂ vapor processes relying on deep vacuum, aerosolization, ionization and plasma generation, alone or in combination, where the difficulties discussed in this publication have not presented themselves. They will not be treated further within.

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